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Mutations in the class I-like major histocompatibility complex gene called HFE are associated with hereditary hemochromatosis (HHC), a disorder of excessive iron uptake. We screened DNA samples from patients with familial Alzheimer disease (FAD) (n = 26), adults with Down syndrome (DS) (n = 50), and older (n = 41) and younger (n = 52) healthy normal individuals, for two HHC point mutations-C282Y and H63D. Because the apolipoprotein E (ApoE) E4 allele is a risk factor for AD and possibly also for dementia of the AD type in DS, DNA samples were also ApoE genotyped. Chi-squared analyses were interpreted at the 0.05 level of significance without Bonferroni corrections. In the pooled healthy normal individuals, C282Y was negatively associated with ApoE E4, an effect also apparent in individuals with DS but not with FAD. Relative to older normals, ApoE E4 was overrepresented in both males and females with FAD, consistent with ApoE E4 being a risk factor for AD; HFE mutations were overrepresented in males and underrepresented in females with FAD. Strong gender effects on the distribution of HFE mutations were apparent in comparisons among ApoE E4 negative individuals in the FAD and healthy normal groups (P < 0.002). Our findings are consistent with the proposition that among ApoE E4 negative individuals HFE mutations are predisposing to FAD in males but are somewhat protective in females. Further, ApoE E4 effects in our FAD group are strongest in females lacking HFE mutations. Relative to younger normals there was a tendency for ApoE E4 and H63D to be overrepresented in males and underrepresented in females with DS. The possibility that HFE mutations are important new genetic risk factors for AD should be pursued further., (Copyright 2000 Wiley-Liss, Inc.)

Ferritin is a ubiquitous protein which plays a major role in iron sequestration, detoxification and storage. In this paper we highlight the role of ferritin in iron homeostasis and describe factors and diseases that affect its expression. We also describe new studies which further characterize the structure and expression of a novel form of ferritin heavy (H) chain mRNA that was identified in brain and discuss possible implications of these findings. Human fetal and adult brain cDNA libraries previously were screened with cDNA for well-characterized liver ferritin H. In addition to 'liver-like' brain ferritin H cDNA, novel ferritin H cDNAs with an additional 279 nucleotide sequence at the 3'untranslated region (UTR) were identified in both libraries (see refs. 1 and 2; Dhar, M., Chauthaiwale, V., and Joshi, J. G., Gene, 1993, 126, 275 and Dhar, M., and Joshi, J. G., J. Neurochem., 1993, 61, 2140). However, relative to liver ferritin H cDNA, these novel cDNAs were incomplete at their 5'ends [see ref. 3; Joshi, J. G., Fleming, J. T., Dhar, M. S., and Chauthaiwale, V., J. Neurol Sci., 1995, 134, (Suppl.), 52]. In the present paper, by sequencing of cDNAs using reverse transcriptase polymerase chain reaction, we show that the 279 nt 3'UTR sequence, a coding sequence identical to that in human liver ferritin H, and a full-length 5'UTR that includes one mRNA regulatory iron-response element sequence, co-exist in at least one species of ferritin H transcript in six normal human adult and six late-onset, sporadic Alzheimer disease (AD) brains. This sequence is the same in the normal and AD brains. Dot-blot analysis of poly A+ RNAs from different human tissues indicates that relative to the coding sequence of ferritin H, expression of the 279 nt 3'UTR sequence varies among different tissues, is highest in the adult brain, and is very low in fetal brain. In normal adult hippocampus, ferritin H RNA with the novel 279 nt sequence localizes strongly to small non-neuronal cells, capillary endothelial cells, and to selected populations of neurons (granule cells of the dentate gyrus). Significant homology was observed between a region in the 279 nt 3'UTR segment of ferritin H RNA and the 3'UTR of cyclooxygenase-2 mRNA (an inducible iron-containing enzyme involved in prostaglandin synthesis). Possible functions for ferritin H protein derived from the novel message and for the elongated 3'UTR and 5'UTR are discussed.

Missense mutations in the presenilin 2 (PS-2) gene on chromosome 1 were sought by direct nucleotide sequence analysis of the open reading frame of 60 pedigrees with familial Alzheimer's disease (FAD). In the majority of these pedigrees, PS-1 and beta-amyloid precursor protein (beta APP) gene mutations had been excluded. While no additional PS-2 pathogenic mutations were detected, four silent nucleotide substitutions and alternative splicing of nucleotides 1338-1340 (Glu325) were observed. Analysis of additional members of a pedigree known to segregate a Met239Val mutation in PS-2 revealed that the age of onset of symptoms is highly variable (range 45-88 years). This variability is not attributable to differences in ApoE genotypes. These results suggest (i) that, in contrast to mutations in PS-1, mutations in PS-2 are a relatively rare cause of FAD; (ii) that other genetic or environmental factor modify the AD phenotype associated with PS-2 mutations; and (iii) that still other FAD susceptibility genes remain to be identified.

Forty-one participants representing diverse professional back-grounds attended a workshop on genetic predictive testing for familial Alzheimer's disease (FAD) on January 23, 1993 at Surrey Place Centre in Toronto, Canada. Rapidly emerging molecular genetic findings in AD indicate that predictive testing is now technologically feasible for selected individuals, although defining eligibility criteria remains problematic. Legal, ethical, biomedical, and psychosocial issues related to establishing predictive testing programs for AD were discussed at the workshop. This article reflects these discussions, provides the current biomedical background for them and examines the Huntington's disease (HD) predictive testing experience. Observations concerning molecular genetic predictive testing for AD in light of its genetic heterogeneity and clinical characteristics, such as usual later age of onset than HD, are presented. It is proposed that predictive testing for AD can now be cautiously offered in a research setting primarily according to the recommendations contained within the Ethical Issues Policy Statement on Huntington's Disease Molecular Genetics Predictive Test. However, in their application to AD, some points in the statement are considered to require emphasis, modification, or currently to be of uncertain applicability. This represents an initial step in an on-going process of debate concerning AD that will be required as new advances occur in genetic and clinical research and in bioethics.

We have further characterized an unusual 22p+ marker chromosome with a double nucleolus organizer region (dNOR) previously identified in a family with late-onset dementia of the Alzheimer type. G-banding and morphology of the marker's q arm were typically normal. However, the p+ arm had a terminal cytological satellite and a GT-positive region at the midpoint. Standard C-banding documented 2 C-positive regions: one was associated with the primary centromere; the other, which was at the midpoint of the p arm, was not associated with a constriction. With replication-banding, there was a darkly staining region in the middle of the p+ arm that resembled the pericentromeric region of a chromosome 21 or 22. Fluorescence in situ hybridization with pXlr 101, a probe recognizing the full repeating unit of rDNA, indicated that the marker had an unusually larger rDNA region; with pU 1.2, a probe recognizing the human rDNA promoter, the signal was a doublet. The marker had 2 signals with a beta-satellite probe, and a second signal in addition to that present at the primary centromere under low stringency with alpha-satellite probes and a classic satellite probe. Immunostaining of chromosome spreads after R-banding and ultraviolet (UV) denaturation showed that the major portion of the marker's p arm was highly methylated.

Probable Alzheimer disease (AD) is described in 79-year-old male twins with monozygosity confirmed by DNA examination. The first twin to be affected began to show signs of intellectual deterioration at age 70. In the other, onset was at age 72. Four of their living sibs (current age range = 75-92) are also suspected to have AD. The possible roles of genetic and environmental factors in the development of AD in this sibship are discussed.

We present prospective clinical and neuropathologic details of a pedigree segregating familial Alzheimer's disease (FAD) associated with a mutation (G----A substitution) at nucleotide 2149 in exon 17 of the amyloid precursor protein (APP) gene. This mutation, which is predicted to cause the missense substitution of isoleucine for valine at codon 717 of APP, cosegregated perfectly with the FAD trait (lod score = 3.49 at theta = 0.00). The earliest clinical manifestations of the disease relate to deficits in memory function, cognitive processing speed, and attention to complex cognitive sets. These changes occurred in the absence of changes in nonmemory language and visuospatial functions. The neuropathologic features of FAD associated with the APP717 mutation in this family include severe neuronal loss, abundant neurofibrillary tangles, amyloid plaques, and amyloid angiopathy. These results provide independent confirmation that mutations in the APP gene are linked to the FAD trait in some families.

A family with a multigenerational history of proven or suspected early-onset Alzheimer's disease (AD) consistent with autosomal-dominant inheritance is described. To date, the pedigree comprises five generations in which there are 13 known affected individuals. The mean age of onset of cognitive deficits in those for whom data are available (n = 11) is 47.6 (s.d. 3.0) years and the mean age of death (n = 10) is 58.8 (s.d. 4.0) years. The variability in the extent and quality of available data illustrates the diagnostic difficulties encountered in ascertaining such an extended pedigree, and the need for caution in interpreting the evidence.

Altered chromatin conformation and increased amounts of aluminum have been observed in the brains of patients with Alzheimer disease. These factors have been shown to affect gene regulation. In this report, we describe how these changes may selectively alter the pool size of the human light chain neurofilament gene and play a fundamental role in the expression of this disease.

We have identified 2 sisters with probable dementia of the Alzheimer type who have an unusual 22-derived marker chromosome with a greatly elongated short arm containing 2 well-separated nucleolus organizer regions. A marker chromosome similar in appearance is uncommon in the general population. Eleven of 24 of their biological relatives were also found to have the marker. The known pedigree of this family encompasses 6 generations in 2 of which there is evidence of 10 cases of dementia of the Alzheimer type. The average age-at-onset of dementia is 65.8 +/- 5.5 years; the average age-at-death among those apparently affected is 74.9 +/- 8.3 years. A new model for the estimation of risk was applied to the family data. Persons in this family with the marker were found to be 4 times more likely to develop dementia than those without the marker, the 95% confidence interval for this risk being 1-50. The probability that the association of dementia and the marker is due to chance alone is .05 (1 in 20).

The technique of in situ hybridization with tritiated RNA probes was used to study the expression of the 68 kDa neurofilament (NF68) gene and the superoxide dismutase-1 (SOD-1) gene in the brains of Alzheimer's disease (AD) patients. Messenger RNA (mRNA) for these proteins was localized and quantified in single cells of formalin-fixed, paraffin-embedded sections of 4 pairs of AD and Huntington's disease (HD) brains from patients matched for age at death and autopsy interval. The cerebellar cortex and hippocampal CA1 and CA2 regions were compared in these two groups of subjects, since in AD the CA2 region of the hippocampus and the cerebellum have been found to be relatively unaffected by the Alzheimer process in comparison to the hippocampal CA1 region. The amount of NF68 mRNA was reduced by approximately 50% in pyramidal cells of both the CA1 and CA2 of AD hippocampus (P less than 0.001), and by 15% in the Purkinje cells of AD cerebellum (P less than 0.05) relative to that of the HD individuals. SOD-1 mRNA was reduced by about 22% in the CA1 of AD brains (P less than 0.001) with no corresponding reduction in the CA2, and by only 5% in the AD cerebellum (P greater than 0.5). The paired design of the study suggests that these results are not simply attributable to the effects of autopsy interval or the agonal process in each patient's death.

Alzheimer's disease, a fatal neurodegenerative disorder of unknown aetiology, is usually considered to be a single disorder because of the general uniformity of the disease phenotype. Two recent genetic linkage studies revealed co-segregation of familial Alzheimer disease with the D21S1/S11 and D21S16 loci on chromosome 21. But two other studies, one of predominantly multiplex kindreds with a late age-of-onset, the other of a cadre of kindreds with a unique Volga German ethnic origin, found absence of linkage at least to D21S1/S11. So far it has not been possible to discern whether these conflicting reports reflect aetiological heterogeneity, differences in methods of pedigree selection, effects of confounding variables in the analysis (for example, diagnostic errors, assortative matings), or true non-replication. To resolve this issue, we have now examined the inheritance of five polymorphic DNA markers from the proximal long arm of chromosome 21 in a large unselected series of pedigrees with familial Alzheimer's disease. Our data suggest that Alzheimer's disease is not a single entity, but rather results from genetic defects on chromosome 21 and from other genetic or nongenetic factors.

Serum tests of thyroid function were compared in Down syndrome (DS) patients with and without manifestations of Alzheimer disease (AD). Relative to control individuals, DS patients had, overall, lower mean total T4 (P = 0.070) and T3f (P = 0.015), higher T3U (P = 0.013) and TSH (P = 0.020), no difference in free T4, and higher thyroid antithyroglobulin (ATA) (P = 0.033) and antimicrosomal autoantibody (AMA) titres (P = 0.0097). Similar trends were apparent in DS males and females, and in DS patients off all drugs. In an analysis of case/control pairs with corrections for age and sex, DS patients with AD manifestations (n = 9) had significantly lower T3 (P = 0.029) and higher AMA (P = 0.043) than paired control individuals, whereas DS patients without AD manifestations (n = 20) had significantly lower T3 (P = 0.013) but higher ATA (P = 0.0065). T3 was significantly lower in the DS patients with AD manifestations than in the unaffected (P = 0.0013). These data suggest that autoimmune thyroiditis associated with a mild "subclinical" form of hypothyroidism is common in adult DS patients and more pronounced in patients with AD manifestations than in those without. This "subclinical" hypothyroidism may contribute to cognitive deficits in ageing DS patients.

The activities of red blood cell enzymes that scavenge the superoxide radical and hydrogen peroxide were measured in severely to profoundly retarded adult Down syndrome (DS) patients with and without manifestations of Alzheimer disease (AD), and control individuals matched for sex, age, and time of blood sampling. Cu,Zn superoxide dismutase (SOD-1) and glutathione peroxidase (GSHPx) activities were significantly elevated (1.39-fold and 1.24-fold, respectively) in DS individuals without AD. When an adjustment was made for the SOD gene dosage effect, DS patients with AD manifestations had significantly lower SOD levels than the matched control individuals. In contrast, DS patients with and without AD had a similar elevation in GSHPx (an adaptive phenomenon). The mean catalase (CAT) activity was no different in DS and control individuals; however, in a paired regression analysis, DS patients without AD had marginally lower CAT activity than control individuals, whereas DS patients with AD had slightly but not significantly higher CAT activity. Thus, AD manifestations in this DS population are associated with changes in the red cell oxygen scavenging processes.